Nitric oxide influences intramuscular signaling that affects skeletal muscle glucose uptake during exercise. The role of the main NO-producing enzyme isoform activated during skeletal muscle contraction, neuronal nitric oxide synthase-μ (nNOSμ), in modulating glucose uptake has not been investigated in a physiological exercise model. In this study, conscious and unrestrained chronically catheterized nNOSμ+/+ and nNOSμ−/− mice either remained at rest or ran on a treadmill at 17 m/min for 30 min. Both groups of mice demonstrated similar exercise capacity during a maximal exercise test to exhaustion (17.7 ± 0.6 vs. 15.9 ± 0.9 min for nNOSμ+/+ and nNOSμ−/−, respectively, P > 0.05). Resting and exercise blood glucose levels were comparable between the genotypes. Very low levels of NOS activity were detected in skeletal muscle from nNOSμ−/− mice, and exercise increased NOS activity only in nNOSμ+/+ mice (4.4 ± 0.3 to 5.2 ± 0.4 pmol·mg−1·min−1, P < 0.05). Exercise significantly increased glucose uptake in gastrocnemius muscle (5- to 7-fold) and, surprisingly, more so in nNOSμ−/− than in nNOSμ+/+ mice ( P < 0.05). This is in parallel with a greater increase in AMPK phosphorylation during exercise in nNOSμ−/− mice. In conclusion, nNOSμ is not essential for skeletal muscle glucose uptake during exercise, and the higher skeletal muscle glucose uptake during exercise in nNOSμ−/− mice may be due to compensatory increases in AMPK activation.